A schematic drawing of a conventional reflection confocal microscope is shown in FIG. 1. Laser light from laser 1 is focussed onto mechanical pinhole 3 by microscope objective 2. The expression "mechanical pinhole" in this specification means a conventional pinhole in a sheet which is typically of metal. The expanding light beam from pinhole 3 is collimated by lens 4 before passing through polarizing beam splitter 8, which polarizes the beam, and quarter waveplate 7, which makes the beam circularly polarized. The beam is then focussed into a diffraction limited spot on object 6 by high quality microscope objective 5. Light reflected and scattered by object 6 is collected by objective 5 which collimates the beam. Light reflected by object 6 remains circularly polarized so that when it passes back through quarter waveplate 7 it is again linearly polarized, but in the direction perpendicular to the original polarized beam. The polarization of light scattered by object 6 being random, will be unaffected by quarter waveplate 7. The light reflected by object 6, with its polarization now rotated by 90 degrees, is redirected by polarizing beam splitter B. A portion of the light scattered by object 6 will also be redirected by polarizing beam splitter 8. The redirected light is focussed onto detector pinhole 10 by imaging element 9. A detector 11 measures the amount of light that passes through the pinhole 10. Object 6 is scanned mechanically in the x, y and z directions by stage 12.
The basis of operation of a reflection confocal microscope can be seen by examination of FIG. 2 which is a schematic drawing of a simplified confocal microscope arrangement. A mechanical pinhole point source of light 15 is imaged onto object 17 by a high quality optical element 16. The illuminating pinhole size 15 is chosen such that light striking object 17 forms a diffraction limited spot pattern whose size is determined by the wavelength of light and the characteristics of high quality optical element 16. The light reflected and scattered by the surface is collected by the high quality optical element 16 and redirected by beam splitter 13 onto a pinhole detector 14. For maximum resolution the size of the pinhole at detector 14 is chosen to be slightly smaller than the first minimum of the diffraction limited spot imaged onto it.
The confocal arrangement described above in FIG. 2 results in a resolution gain of approximately 0.4 over that of conventional microscopes. By using an annulus, this resolution gain is increased to approximately 0.7 over that of a conventional microscope. Additionally, the confocal microscope has a much reduced depth of field, when compared to that of conventional microscopes, which enables out-of-focus information to be removed from the image. This enables rough, curved or partially transparent surfaces to be properly imaged.
In order to obtain an image of an object, the object (or the microscope) is scanned in x, y and z directions with the maximum signal during a z scan being chosen as the intensity at the x, y position. For partially transparent objects, such as biological cells, three dimensional information can be extracted. There is no limit to the size of the area that can be imaged without compromising the resolution. It should be noted that the signal from a confocal microscope is readily amenable to electronic image enhancement.
Mechanical pinholes are susceptible to dirt lodging in the aperture. Even the smallest amount of dirt in a mechanical pinhole in a confocal microscope creates a problem as the resultant light field is no longer circularly symmetrical and aberrations are introduced. Further, slight misalignment of a mechanical pinhole or any other element in a conventional confocal microscope causes asymmetric intensity distribution of the light beam emerging from the mechanical pinhole again causing aberrations.